1. Field of the Invention
The present invention relates generally to the processing of volumetric capillary cytometry data for the purpose of counting and characterizing cells or cell constituents in a volume of material.
2. Description of Related Art
The optical analysis of biological specimens, such as blood, has widespread applications. There are many computer controlled instruments in the market for providing such analysis, including flow cytometers, automated blood cell analyzers and blood cell classifiers.
As described in the above cross-referenced application, a volumetric cytometry instrument has many advantages. In particular, the amount of blood being analyzed is controllable, the handling of the blood is reduced, and analyzed samples of blood can be stored for further processing.
However, the processing of blood samples in a volumetric system raises a number of problems. Particularly if counting of blood cells is required, it is necessary to analyze the entire volume in the capillary or other container which holds the material to be analyzed. Blood cells on the side of a container may be difficult to detect, if the analysis instrument is not calibrated for the precise dimensions of the container. For instance, in Kamentsky, U.S. Pat. No. 5,072,382, samples of blood were applied to a slide. A region to be analyzed was defined by synchronization pulses in the scanning apparatus. (See column 14, lines 49-63 of Kamentsky). Synchronization pulses in the scanning mechanism cannot be precisely aligned with a container such as a capillary or cuvette for a blood sample because of the variations in the shapes of such containers, and variations in the alignment of mounts for the containers in the scanning mechanism. It will be appreciated that the ability to precisely mount and manufacture containers is quite advanced. However, the scanning of containers for the purposes of processing cells may require resolution on the micron scale.
An additional difficulty arises because of the characteristics of dyes used to mark target cells. For instance, when analyzing cells for the presence of specific antibodies, it is common to tag the cells with dyes which fluoresce with a particular spectrum in response to an excitation beam. If more than one antibody is to be detected, more than one dye is used. However, the fluorescence spectrums of various dyes may overlap. Thus, it is difficult to fully process the information in detected fluorescence generated by plural dyes with overlapping spectra.
Furthermore, when it is necessary to count a particular number of target cells within a volume, to achieve a statistically valid count, a relatively large sample must be used. A large sample of blood, when it is scanned on the micron scale, can generate very large amounts of data. It is important for practical analysis machines that the data be processed in a reasonable amount of time. For instance, as described in the above cross-referenced application, the sample can scan for the presence of two dyes with overlapping spectra, with two channels of data. Each channel of data includes information relevant to both dyes. Further, the scan involves about 10,000 lines of 200 pixels each, resulting in 2 million samples per channel, which for 2 bytes per sample in 2 channels amounts to a total of 8 megabytes of raw data.
Furthermore, the fluorescence monitoring techniques are susceptible to a low signal-to-noise ratio. Thus, it is important to be able to process these large amounts of data with high background noise to accurately characterize and identify target cells within the volume, particularly when unbound antibody is present.
Accordingly, it is desirable to provide a method and apparatus for processing data from a volumetric cytometry system which is robust and accurate. Further, the system should be relatively fast and operate in a system having a relatively low memory requirement.